The invention relates to cassettes for the expression of storable proteins in plants, especially of single-chain antibody fragments in transgenic tobacco or pea plants.
Areas of application of the invention are biotechnology, medicine (diagnostics and therapy), food and plant monitoring, as well as agriculture.
Established and reliable methods of gene cloning and gene technology and the newest developments in the technology of transgenic plants make possible further advances in plant biotechnology. Plant parts can more and more serve as production sites for materials, which are otherwise difficult to obtain. Thus, immunoglobulin has been expressed successfully in the leaves of transgenic tobacco plants. The results obtained there lie between 0.1 and 1.3% of the entire soluble protein of the leaf. The expression was either cytoplasmatic or took place in apoplasts of plant cells.
By attaching a signal for retention in the endoplasmic reticulum (ER) to a single-chain antibody gene (scFv-Gen), the antibody can be fixed in this special compartment by leaf cells of transgenic plants. This fixation leads to an increase in the expression rate of single-chain antibody fragments in leaves of transgenic plants to 4.8% of the total soluble protein (Artssenko et al., Plant J. 8, 745-750 (1995)). These findings were confirmed by others in principle, although the absolute expression values were not attained.
Further work relates to the specific expression of gene production in plant storage organs, especially in seeds. With the aid of a seed-specific promoter, single-chain antibody fragments could be expressed in a stable manner up to 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio/Technology 10, 1090-1094 (1995)).
In spite of this progress, the expression rates achieved in plants were previously too low to base a plant biotechnological production of the desired materials on them.
It was therefore an object of the invention to place the seed-specific expression in transgenic plants on a basis suitable for material production. It was a further objective to achieve a biological basis for the use of a simple and manageable harvesting and processing technology, above all to assure that the gene product formed in the plant is preserved in a stable manner during the time between the direct harvest and the following extraction and purification steps in quantity and desired activity at ambient temperatures, without refrigeration.
The inventive expression cassettes contain a promoter (preferably a constitutive promoter such as the CaMV 35S promoter or a seed-specific promoter), the LeB4 signal peptide, the gene to be expressed and an ER retention signal.